This is a fast-track proposal. Phase I: As more molecular targets for diseases are identified and the promise of personalized medicine delivers more effective therapeutics, increasingly sensitive protein bioassays will be needed to take advantage of these trends. These improved assays will allow for: (1) ability to evaluate and validate new drug targets, (2) better match patients to new therapies, (3) accelerate drug approval, (4) enable basic understanding of biological functions, and (5) aid in the diagnosis of at risk patients. With this Phase I proposal, extremely sensitive Erenna[unreadable] bioassays against the human and mouse vascular endothelial growth factor (VEGF) will be developed. These assays will be able to detect VEGF at low pg/ml levels and will require as little as 5 ul of material. These assays will be developed for use with cell lysates, serum and tissue samples. Preliminary analysis will determine the level of assay-to-assay, machine-to-machine and lab-to-lab variability. Furthermore, the assays will be used to assess tumor and normal breast cancer tissue both in humans and mouse models of breast cancer. These studies will be used to compare a mouse model to human disease and will also provide preliminary data assessing the level of VEGF in tumors at different stages of disease progression. Phase II: More and higher sensitivity protein biomarker assays will provide new tools for: (1) the evaluation of new drug targets, (2) matching patients to new therapies, (3) acceleration of drug approval, (4) enabling of basic research, and (5) providing new diagnostics. With this Phase II proposal, extremely sensitive Erenna[unreadable] bioassays against the human and mouse Akt1, GSK3-beta, JNK1, JNK2 and ERK 2 will be developed. These assays will be able to detect the proteins at sub pg/ml levels and will require as little as 5 ul of material. These assays will be developed for use with cell lysates, serum and tissue samples. They, along with the Erenna[unreadable] VEGF assays, will be validated for specificity, linearity, range, detection limits, accuracy, robustness and precision in diseased and normal patient and mouse samples over a 6 month period. The studies will examine the correlation of tumor biomarker levels to serum levels, as well as to compare biomarker levels in mouse models to the comparable human disease tissue. Correlation studies comparing biomarker levels to disease state will be carried out. Finally, for use with human serum samples, even greater sensitivity Erenna[unreadable] Akt1, GSK3-beta, JNK1, JNK2 and ERK2 bioassays will be developed and validated.